Increase In Glomerular Permeability Research Articles

Sera From Patients With Minimal Change Disease Increase Endothelial Permeability to Sodium

Edema is the main symptom of nephrotic syndrome associated to minimal change disease (MCD). Besides the increase in glomerular permeability, possibly induced by a circulating permeability factor,1 an increase in systemic vascular permeability could participate in the constitution of edema.

Glomerular permeability is not affected by heparan sulfate glycosaminoglycan deficiency in zebrafish embryos

Proteinuria develops when specific components in the glomerular filtration barrier have impaired function. Although the precise components involved in maintaining this barrier have not been fully identified, heparan sulfate proteoglycans are believed to play an essential role in maintaining glomerular filtration. Although in situ studies have shown that a loss of heparan sulfate glycosaminoglycans increases the permeability of the glomerular filtration barrier, recent studies using experimental models have shown that podocyte-specific deletion of heparan sulfate glycosaminoglycan assembly does not lead to proteinuria. However, tubular reabsorption of leaked proteins might have masked an increase in glomerular permeability in these models. Furthermore, not only podocytes but also glomerular endothelial cells are involved in heparan sulfate synthesis in the glomerular filtration barrier. Therefore, we investigated the effect of a global heparan sulfate glycosaminoglycan deficiency on glomerular permeability. We used a zebrafish embryo model carrying a homozygous germline mutation in the ext2 gene. Glomerular permeability was assessed with a quantitative dextran tracer injection method. In this model, we accounted for tubular reabsorption. Loss of anionic sites in the glomerular basement membrane was measured using polyethyleneimine staining. Although mutant animals had significantly fewer negatively charged areas in the glomerular basement membrane, glomerular permeability was unaffected. Moreover, heparan sulfate glycosaminoglycan-deficient embryos had morphologically intact podocyte foot processes. Glomerular filtration remains fully functional despite a global reduction of heparan sulfate.

Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the glomerulus and reabsorbed in the tubular system up until the absorption limit is reached. Early increases in pathological albumin filtration may, thus, be missed by analyzing albuminuria. Therefore, the use of tracers to test glomerular permselectivity appears advantageous. Fluorescently labeled tracer fluorescein isothiocyanate (FITC)-polysucrose (i.e., FITC-Ficoll), can be used to study glomerular permselectivity. FITC-polysucrose molecules are freely filtered by the glomerulus but not reabsorbed in the tubular system. In mice and rats, FITC-polysucrose has been investigated in models of glomerular permeability by using technically complex procedures (i.e., radioactive measurements, high-performance liquid chromatography [HPLC], gel filtration). We have modified and facilitated a FITC-polysucrose tracer-based protocol to test early and small increases in glomerular permeability to FITC-polysucrose 70 (size of albumin) in mice. This method allows repetitive urine analyses with small urine volumes (5 µL). This protocol contains information on how the tracer FITC-polysucrose 70 is applied intravenously and urine is collected via a simple urinary catheter. Urine is analyzed via a fluorescence plate reader and normalized to a urine concentration marker (creatinine), thereby avoiding technically complex procedures.

Sustained, delayed, and small increments in glomerular permeability to macromolecules during systemic ET-1 infusion mediated via the ETA receptor.

Emerging evidence indicates that endogenous production of endothelin (ET)-1, a 21-amino acid peptide vasoconstrictor, plays an important role in proteinuric kidney disease. Previous studies in rats have shown that chronic administration of ET-1 leads to increased glomerular albumin leakage. The underlying mechanisms are, however, currently not known. Here, we used size-exclusion chromatography to measure glomerular sieving coefficients for neutral FITC-Ficoll (molecular Stokes-Einstein radius: 15-80 Å, molecular weight: 70 kDa/400 kDa) in anesthetized male Sprague-Dawley rats (n = 12) at baseline and at 5, 15, 30, and 60 min after intravenous administration of ET-1. In separate experiments, ET-1 was given together with the selective ET type A (ETA) or ET type B (ETB) receptor antagonists JKC-301 and BQ-788, respectively. At both 15 and 30 min postadministration, the glomerular sieving coefficient for macromolecular Ficoll (70 Å) was significantly increased to 4.4 × 10-5 ± 0.7 × 10-5 (P = 0.024) and 4.5 × 10-5 ± 0.8 × 10-5 (P = 0.007), respectively, compared with baseline (2.2 × 10-5 ± 0.4 ×10-5). Decreased urine production after ET-1 prevented the use of higher doses of ET-1. Data analysis using the two-pore model indicated changes in large-pore permeability after ET-1, with no changes in the small-pore pathway. Administration of ETA blocker abrogated the permeability changes induced by ET-1 at 30 min, whereas blockade of ETB receptors was ineffective. Mean arterial pressure was only significantly increased at 60 min, being 123 ± 4 mmHg compared with 111 ± 2 mmHg at baseline (P = 0.02). We conclude that ET-1 evoked small, delayed, and sustained increases in glomerular permeability, mediated via the ETA receptor.

Nitric oxide synthase inhibition causes acute increases in glomerular permeability in vivo, dependent upon reactive oxygen species.

There is increasing evidence that the permeability of the glomerular filtration barrier (GFB) is partly regulated by a balance between the bioavailability of nitric oxide (NO) and that of reactive oxygen species (ROS). It has been postulated that normal or moderately elevated NO levels protect the GFB from permeability increases, whereas ROS, through reducing the bioavailability of NO, have the opposite effect. We tested the tentative antagonism between NO and ROS on glomerular permeability in anaesthetized Wistar rats, in which the left ureter was cannulated for urine collection while simultaneously blood access was achieved. Rats were systemically infused with either l-NAME or l-NAME together with the superoxide scavenger Tempol, or together with l-arginine or the NO-donor DEA-NONOate, or the cGMP agonist 8-bromo-cGMP. To measure glomerular sieving coefficients (theta, θ) to Ficoll, rats were infused with FITC-Ficoll 70/400 (mol/radius 10-80 Å). Plasma and urine samples were analyzed by high-performance size-exclusion chromatography (HPSEC) for determination of θ for Ficoll repeatedly during up to 2 h. l-NAME increased θ for Ficoll70Å from 2.27 ± 1.30 × 10-5 to 8.46 ± 2.06 × 10-5 (n = 6, P < 0.001) in 15 min. Tempol abrogated these increases in glomerular permeability and an inhibition was also observed with l-arginine and with 8-bromo-cGMP. In conclusion, acute NO synthase inhibition in vivo by l-NAME caused rapid increases in glomerular permeability, which could be reversed by either an ROS antagonist or by activating the guanylyl cyclase-cGMP pathway. The data strongly suggest a protective effect of NO in maintaining normal glomerular permeability in vivo.

The Effect Of An Active Versus Inactive Lifestyle On Renal Response To Exercise-induced Dehydration

PURPOSE: Exercise by itself as well as exercise-induced dehydration cause a decrease in renal blood flow and an increase in glomerular permeability, leading to a decrement in renal function and temporarily renal damage. An active lifestyle may precondition the kidneys, resulting in attenuated decrease in kidney function and a reduction in kidney damage in response to exercise and dehydration determined as body weight loss. Therefore, we examined the differences in renal responses to exercise-induced dehydration between healthy young men with a physically active versus inactive lifestyle. METHODS: A total of 12 active and 6 inactive healthy male participants (23±3 years, 23.9±3.6 kg/m2, eGFR: 111.0±12.5 mL/min) were included, based on a physical activity questionnaire. Participants performed an incremental exercise test to determine physical fitness and maximum heart rate (HRmax). On a separate day, a submaximal exercise test was performed on 80% of the individual HRmax, until 3% dehydration. Blood and urine samples were taken pre- and post-exercise to assess renal function (estimated glomerular filtration ratio; eGFR) and renal damage (urinary albumin). RESULTS: Maximum oxygen uptake (VO2max) was higher in the active (59.7±8.6 mL/min/kg) compared to the inactive group (49.1±7.3 mL/min/kg; p=0.018). All subjects cycled on a comparable exercise intensity (79±2% HRmax), resulting in a body mass loss of 3.2±0.7% and 2.5±0.4% for the active and inactive group, respectively (p=0.013). After exercise, a significant decrease in eGFR was found for the active (17.4±10.2 mL/min; p=0.007) and inactive group (21.7±17.8 mL/min; p=0.05), which did not differ between groups (p=0.60). Moreover, 33.3% of the active subjects and 50% of the inactive subjects had a post-exercise eGFR<90 mL/min, which indicates a decrease in renal function. Furthermore, we found a significant increase in urinary albumin concentration in the active (24.6±30.9 mg/L; p=0.011) and inactive group (33.6±27.5 mg/L; p=0.016), which did not differ between groups (p=0.55). CONCLUSION: In short, exercise induces a decrease in renal function and an increase in urinary albumin concentration, suggesting temporary renal damage of the glomerulus or proximal tubulus. However, these changes do not differ between physically active and inactive subjects.

Acute reactive oxygen species (ROS)-dependent effects of IL-1β, TNF-α, and IL-6 on the glomerular filtration barrier (GFB) in vivo.

This study was performed to investigate the immediate actions of the proinflammatory cytokines IL-1β, TNF-α, and IL-6 on the permeability of the glomerular filtration barrier (GFB) in rats and to test whether these actions are dependent upon the release of reactive oxygen species (ROS). In anesthetized rats, blood access was achieved and the left ureter was cannulated for urine collection. Rats were continuously infused intravenously with either IL-1β (0.4 and 2 μg·kg(-1)·h(-1)), TNF-α (0.4 and 2 μg·kg(-1)·h(-1)), or IL-6 (4 and 8 μg·kg(-1)·h(-1)), together with polydisperse FITC-Ficoll-70/400 and inulin for 1 h. Plasma and urine samples were analyzed by high performance size exclusion chromatography (HPSEC) for determination of glomerular sieving coefficients (θ). The glomerular filtration rate (GFR) was also assessed (51Cr-EDTA). In separate experiments, the superoxide scavenger tempol (30 mg·kg(-1)·h(-1)) was given before and during cytokine infusions. IL-1β and TNF-α caused rapid, partly reversible increases in glomerular permeability to large molecules (Ficoll50-80Å), peaking at 5-30 min, while IL-6 caused a more gradual increase in permeability, leveling off at 60 min. Tempol almost completely abrogated the glomerular permeability effects of the cytokines infused. In conclusion IL-1β, TNF-α, and IL-6, when infused systemically, caused immediate and partly reversible increases in glomerular permeability, which could be inhibited by the superoxide scavenger tempol, suggesting an important role of ROS in acute cytokine-induced permeability changes in the GFB.

Renal Sodium Handling in Children with Nephrotic Syndrome

Background and Objectives: Neprhrotic Syndrome (NS) is a predictable complex with severe, prolonged increase in glomerular permeability for protein leading to hypo-albuminemia. Edema is the other major abnormality; but the underlying mechanism is incompletely resolved. Again during edema formation stage, some of these children express clinical symptoms of hypovolemia, while others do not. The purpose of the study was to identify the NS-children with hypovolemia by measuring the parameters of renal function in respect to their specificity and easy availability.&#x0D; Methods: We studied renal function in 17 children with NS in full blown nephrosis who had come to the Nephrology-Follow up Clinic or the OPD in Dhaka Shishu (Children) Hospital (DSH) and got admitted into the hospital as nephrotic patients with relapses or 1st episodes. The period of study was July 2001 to December 2001 for total period of 6 months. Findings were related to presence or absence of symptoms suggestive of hypovolemia, and were compared to results of similar studies in the same children in remission.&#x0D; Results: Nine children presented with hypovolemic symptoms, and 8 without such symptoms. Both groups displayed severe proteinuria, hypo-albuminemia and edema. Twelve (70.5%) children showed higher blood pressure values in comparision to those of remission. Symptomatic patients showed tendency for a low glomerular filtration rate (GFR), and significantly impaired urine dilution, lower urine-sodium (UNa/Ucr), decreased fractional sodium excretion (FENa), and elevated sodium-potassium exchange quotient, UK/(UK+UNa). In the non-symptomatic patients, these parameters were normal.&#x0D; Conclusions: Among parameters of renal functions,UK/(UK+UNa)-sodium-potassium exchange quotient was found to be the most specific - higher values in hypovolemic patients and lower in patients with stable edema, and in remission too.&#x0D; Ibrahim Cardiac Med J 2013; 3(1&amp;2): 15-20

MTOR inhibition with temsirolimus causes acute increases in glomerular permeability, but inhibits the dynamic permeability actions of puromycin aminonucleoside.

Inhibitors of the mammalian target of rapamycin (mTORi) can produce de novo proteinuria in kidney transplant patients. On the other hand, mTORi has been shown to suppress disease progression in several animal models of kidney disease. In the present study, we investigated whether glomerular permeability can be acutely altered by the mTORi temsirolimus and whether mTORi can affect acute puromycin aminonucleoside (PAN) or angiotensin II (ANG II)-induced glomerular hyperpermeability. In anesthetized Wistar rats, the left ureter was cannulated for urine collection, while simultaneously blood access was achieved. Temsirolimus was administered as a single intravenous dose 30 min before the start of the experiments in animals infused with PAN or ANG II or in nonexposed animals. Polydispersed FITC-Ficoll-70/400 (molecular radius 10-80 Å) and (51)Cr-EDTA infusion was given during the whole experiment. Measurements of Ficoll in plasma and urine were performed sequentially before the temsirolimus injection (baseline) and at 5, 15, 30, 60, and 120 min after the start of the experiments. Urine and plasma samples were analyzed by high-performance size-exclusion chromatography (HPSEC) to assess glomerular sieving coefficients (θ) for Ficoll10-80Å. Temsirolimus per se increased baseline glomerular permeability to Ficoll50-80Å 45 min after its administration, a reactive oxygen species (ROS)-dependent phenomenon. PAN caused a rapid and reversible increase in glomerular permeability, peaking at 5 min, and again at 60-120 min, which could be blocked by the ROS scavenger tempol. mTORi abrogated the second permeability peak induced by PAN. However, it had no effect on the immediate ANG II- or PAN-induced increases in glomerular permeability.

Modification of the relationship between blood pressure and renal albumin permeability by impaired excretory function and diabetes.

In animal models, reduced nephron mass impairs renal arteriolar autoregulation, increasing vulnerability of the remaining nephrons to elevated systemic blood pressure (BP). A feature of the resulting glomerular capillary hypertension is an increase in glomerular permeability. We sought evidence of a similar remnant nephron effect in human chronic kidney disease. In participants from the United States National Health and Nutrition Examination Surveys 1999 to 2010 (N=23 710), we examined the effect of reduced estimated glomerular filtration rate (eGFR) on the relationship between brachial artery BP and albumin permeability. Renal albumin permeability increased exponentially with systolic BP >110 mm Hg, and this association was modified by independent interactions with both excretory impairment and diabetes mellitus. Each 10 mm Hg increase in systolic BP was accompanied by an increase in fractional albumin excretion of 1.10-, 1.11-, 1.17-, 1.22-, and 1.38-fold for participants with eGFR≥90, 90>eGFR≥60, 60>eGFR≥45, 45>eGFR≥30, and eGFR<30 mL/min/1.73 m(2), respectively, adjusted for age, sex, race, antihypertensive use, eGFR category, diabetes mellitus, smoking, history of cardiovascular disease, body mass index, and C-reactive protein. A 10 mm Hg systolic BP increment was associated with increases in fractional albumin excretion of 1.10- and 1.21-fold in nondiabetic and diabetic participants, respectively. Using urine albumin creatinine ratio as an alternative measure of albumin leak in eGFR-adjusted analyses gave the same conclusions. Our findings are consistent with the presence of a remnant nephron effect in human kidney disease. Future trials should consider the nephroprotective benefits of systolic BP lowering in kidney disease populations stratified by eGFR.

Extracellular fetal hemoglobin induces increases in glomerular permeability: inhibition with α1-microglobulin and tempol

Extracellular fetal hemoglobin (HbF) and adult hemoglobin (HbA) are proinflammatory and generate ROS. Increased plasma levels of extracellular HbF have recently been reported to occur in early preeclampsia. α1-Microglobulin (A1M) is a physiological heme-binding protein and radical scavenger that has been shown to counteract vascular permeability increases induced by HbA in the perfused placenta. The present study was performed to investigate whether HbF and HbA will increase glomerular permeability in vivo and to test whether A1M and tempol, a ROS scavenger, can prevent their effects. Anesthetized Wistar rats were continuously infused intravenously with either HbA, HbF, or cyano-inactivated HbF together with FITC-Ficoll-70/400, inulin, and (51)Cr-labeled EDTA for 2 h. Plasma samples and urine samples (left ureter) were taken repeatedly and analyzed by high-performance size exclusion chromatography to assess glomerular sieving coefficients for Ficoll of radius 10-80 Å. In separate experiments, A1M or tempol was given before and during Hb infusions. Extracellular HbF caused rapid, transient increases in glomerular permeability to large Ficoll molecules (50-80Å), contrary to the effects of HbA and cyano-inactivated HbF. For HbF, glomerular sieving coefficients for Ficoll of radius 60Å increased from 3.85 ± 0.85 × 10(-5) to 2.60 ± 0.96 × 10(-4) at 15 min, changes that were abrogated by tempol and reduced by A1M. In conclusion, our data demonstrate that extracellular HbF, infused systemically, can acutely increase glomerular permeability through inducing oxidative stress.

Scavengers of reactive oxygen species, paracalcitol, RhoA, and Rac-1 inhibitors and tacrolimus inhibit angiotensin II-induced actions on glomerular permeability

Systemic infusions of ANG II rapidly induce large, dynamic increases in the permeability of the glomerular filtration barrier (GFB) in rats. After binding to its receptor(s), ANG II generates reactive oxygen species (ROS) and produces Ca²⁺ influx into cells, leading to activation of a plethora of signaling cascades, including, e.g., calcineurin and small GTPases, such as Rac-1 and RhoA. In the present study we sought to interact with some of these cascades to test potential novel antiproteinuric agents. In anesthetized Wistar rats, the left urether was cannulated for urine collection, and blood access was achieved. Rats were infused with ANG II (16 ng·kg⁻¹·min⁻¹) alone, or together with the ROS scavengers tempol or dimethylthiourea (DMTU) or the D-vitamin analog paracalcitol, the RhoA-kinase inhibitor Y-27632, the Rac-1 inhibitor NSC-23766, or the calcineurin inhibitor tacrolimus. FITC-Ficoll-70/400 (mol.radius 10-80 Å) and ⁵¹Cr-EDTA were infused throughout the experiment. Plasma and urine samples were taken during baseline and at 5 and 15 min after the start of the infusions and analyzed by high-performance size-exclusion chromatography for determination of glomerular sieving coefficients (θ) for Ficoll10-80Å. ANG II infusion into rats caused marked increases in glomerular permeability to large Ficoll molecules (Ficoll50-80Å), which were abrogated by the ROS scavenger tempol and partly by DMTU. Paracalcitol, RhoA, and Rac-1 inhibition, and, to some extent tacrolimus, but not prostacyclin, could also inhibit the glomerular permeability actions of ANG II. Our data suggest that cellular ROS generation and active Ca²⁺ signaling are involved in ANG II-induced increases in glomerular permeability.

Rapid, dynamic changes in glomerular permeability to macromolecules during systemic angiotensin II (ANG II) infusion in rats

The actions of systemic angiotensin II (ANG II) infusions on glomerular permeability were investigated in vivo. In anesthetized Wistar rats (250-280 g), the left ureter was cannulated for urine collection, while simultaneously blood access was achieved. Rats were continuously infused intravenously with either of four doses of ANG II ranging from 16 ng·kg(-1)·min(-1) (Lo-ANG II) to 1.82 μg·kg(-1)·min(-1) (Hi-ANG II), and in separate experiments with aldosterone (Aldo; 0.22 mg·kg(-1)·min(-1)), or with the calcium channel blocker nimodipine, or with the Aldo antagonist spironolactone together with a high ANG II dose (910 ng·kg(-1)·min(-1); Hi-Int-ANG II), respectively, and with polydisperse FITC-Ficoll-70/400 (molecular radius 10-80 Å) and (51)Cr-EDTA. Plasma and urine samples were taken at 5, 15, 30, 60, and 120 min and analyzed by high performance size-exclusion chromatography for determination of glomerular sieving coefficients (θ) to Ficoll. Mean arterial pressure (MAP) and glomerular filtration rate (GFR) were also assessed. For ANG II, there was a rapid, marked, partly reversible increase in glomerular permeability (θ) for Ficoll molecules >34 Å in radius, peaking at 5-15 min, which was completely abrogated by the ANG II blocker candesartan but not affected by spironolactone at 15 and 30 min. For Aldo, the response was similar to that found for the lowest dose of ANG II infused. For the two highest ANG II doses given (Hi-Int-ANG II and Hi-ANG II), GFR decreased transiently, concomitant with marked, sustained increases in MAP. Nimodipine completely blocked all hemodynamic ANG II actions, whereas the glomerular permeability response remained unchanged. Thus ANG II directly increased glomerular permeability independently of its hemodynamic actions and largely independently of the concomitant Aldo response. The ANG II-induced increases in glomerular permeability were, according to a two-pore and a log-normal distributed pore model, compatible with an increased number of "large pores" in the glomerular filter, and, to some extent, an increase in the dispersity of the small-pore radius.

Transient and sustained increases in glomerular permeability following ANP infusion in rats

The present study was performed to investigate the effects of systemic atrial natriuretic peptide (ANP) infusion on the glomerular permeability to macromolecules in rats. In anesthetized Wistar rats (250-280 g), the left urether was cannulated for urine collection while simultaneously blood access was achieved. Rats were continuously infused intravenously with ANP [30 ng·kg(-1)·min(-1) (Lo-ANP; n=8) or 800 ng·kg(-1)·min(-1) (Hi-ANP; n=10)] or 0.9% NaCl (SHAM; n=16), respectively, and with polydisperse FITC-Ficoll-70/400 (molecular radius 13-90 Å) and 51Cr-EDTA for 2 h. Plasma and urine samples were taken at 5, 15, 30, 60, and 120 min of ANP infusion and analyzed by high-performance size-exclusion chromatography (HPLC) for determination of glomerular sieving coefficients (θ) for Ficoll. GFR was also assessed (51Cr-EDTA). In Hi-ANP, there was a rapid (within 5 min), but bimodal, increase in glomerular permeability. θ to high-molecular-weight Ficoll thus reached a maximum at 15 min, after which θ returned to near control at 30 min, to again increase moderately at 60 and 120 min. In Lo-ANP, there was also a rapid, reversible increase in glomerular θ, returning to near control at 30 min, followed by just a tendency of a sustained increase in permeability, but with a significant increase in "large-pore" radius. In conclusion, in Hi-ANP there was a rapid increase in glomerular permeability, with an early, partly reversible permeability peak, followed by a (moderate) sustained increase in permeability. In Lo-ANP animals, only the initial permeability peak was evident. In both Lo-ANP and Hi-ANP, the glomerular sieving pattern observed was found to mainly reflect an increase in the number and radius of large pores in the glomerular filter.

Acute hyperglycemia induces rapid, reversible increases in glomerular permeability in nondiabetic rats

This study was performed to investigate the impact of acute hyperglycemia (HG) on the permeability of the normal glomerular filtration barrier in vivo. In anesthetized Wistar rats (250-280 g), the left ureter was catheterized for urine collection, while simultaneously blood access was achieved. Rats received an intravenous (iv) infusion of either 1) hypertonic glucose to maintain blood glucose at 20-25 mM (G; n = 8); 2) hypertonic glucose as in 1) and a RhoA-kinase inhibitor (Y-27632; Rho-G; n = 8); 3) 20% mannitol (MANN; n = 7) or 4) hypertonic (12%) NaCl to maintain plasma crystalloid osmotic pressure (pi(cry)) at approximately 320-325 mosmol/l (NaCl; n = 8) or 5) physiological saline (SHAM; n = 8). FITC-Ficoll 70/400 was infused iv for at least 20 min before termination of the experiments, and plasma and urine were collected to determine the glomerular sieving coefficients (theta) for polydisperse Ficoll (molecular radius 15-80 A) by high-performance size-exclusion chromatography. In G there was a marked increase in for Ficoll(55-80A) at 20 min, which was completely reversible within 60 min and abrogated by a Rho-kinase (ROCK) inhibitor, while glomerular permeability remained unchanged in MANN and NaCl. In conclusion, acute HG caused rapid, reversible increases in for large Ficolls, not related to the concomitant hyperosmolarity, but sensitive to ROCK inhibition. The changes observed were consistent with the formation of an increased number of large pores in the glomerular filter. The sensitivity of the permeability changes to ROCK inhibition strongly indicates that the cytoskeleton of the cells in the glomerular barrier may be involved in these alterations.

Formation of lipid raft redox signalling platforms in glomerular endothelial cells: an early event of homocysteine‐induced glomerular injury

The present study tested the hypothesis that homocysteine (Hcys)-induced ceramide production stimulates lipid rafts (LRs) clustering on the membrane of glomerular endothelial cells (GECs) to form redox signalling platforms by aggregation and activation of NADPH oxidase subunits and thereby enhances superoxide (O2.−) production, leading to glomerular endothelial dysfunction and ultimate injury or sclerosis. Using confocal microscopy, we first demonstrated a co-localization of LR clusters with NADPH oxidase subunits, gp91phox and p47phox in the GECs membrane upon Hcys stimulation. Immunoblot analysis of floated detergent-resistant membrane fractions found that in LR fractions NADPH oxidase subunits gp91phox and p47phox are enriched and that the activity of this enzyme dramatically increased. We also examined the effect of elevated Hcys on the cell monolayer permeability in GECs. It was found that Hcys significantly increased GEC permeability, which was blocked by inhibition of LR redox signalling platform formation. Finally, we found that Hcys-induced enhancement of GEC permeability is associated with the regulation of microtubule stability through these LR-redox platforms. It is concluded that the early injurious effect of Hcys on the glomerular endothelium is associated with the formation of redox signalling platforms via LR clustering, which may lead to increases in glomerular permeability by disruption of microtubule network in GECs.

Loss of size selectivity of the glomerular filtration barrier in rats following laparotomy and muscle trauma

Posttraumatic microalbuminuria may be caused by either charge- or size-selective alterations in the glomerular filtration barrier, or both, and/or to a reduction in proximal tubular protein reabsorption. This study was performed to elucidate the pathophysiology of the increases in glomerular permeability occurring in rats exposed to a laparotomy or to a laparotomy and muscle trauma. In anesthetized Wistar rats (250-280 g), the left ureter was cannulated for urine collection, while simultaneously blood access was achieved. Rats were exposed to trauma by a laparotomy (L; n = 8), or by a combination of L and muscle trauma (MT; L+MT) induced by topical blunt injury of the abdominal muscles bilaterally. After L, muscles were crushed using hemostatic forceps at either 2 x 2 sites ("small" MT; n = 9), or at 2 x 5 sites ("large" MT; n = 9). Sham groups (n = 16), not exposed to a laparotomy, were used as controls. The glomerular sieving coefficients (theta) to polydisperse FITC-Ficoll-70/400 (molecular radius 13-80 A) were determined at 5 or 60 min after L and L+MT, respectively, from plasma and urine samples, and analyzed by high-performance size-exclusion chromatography. A tissue-uptake technique was used to assess theta for (125)I-labeled serum albumin. L, with or without MT, increased theta for Ficoll(55-80A) and albumin rapidly and markedly. Theta-Ficoll(70A) thus increased approximately threefold, and theta for albumin significantly, for all trauma groups. According to the "two-pore model" of glomerular permeability, these changes mainly reflect an increase in the number of large pores in the glomerular filter without any primary changes in the charge-selective properties of the filter.

ETA receptor dependent increases in glomerular permeability in experimentally induced diabetic rats

A glomerular permeability defect occurs early in type 1 diabetes and precedes the onset of albuminuria and renal morphological changes. The aim of the current study was to test the hypothesis that the ETA receptor contributes to increased glomerular albumin permeability (Palb) in a model of type 1 diabetes. Male SD rats (250‐275 g) were given streptozotocin (diabetic, D; i.v., 65 mg/kg) or saline (S). Half of each group received the ETA‐selective antagonist, ABT‐627 (ABT), in drinking water at 5 mg/kg/d (n=6 in all groups). Treatment was begun immediately after confirming plasma glucose &gt;300 mg/dl. Six weeks later, glomeruli were isolated by sieving techniques and Palb determined from the change in glomerular volume induced by exposing glomeruli to oncotic gradients of albumin. Palb was increased in D (0.91 ±0.09 vs. S, 0.05 ±0.01, P&lt;0.01); ABT significantly reduced Palb when compared with D (D+ABT, 0.40 ±0.10, P&lt;0.05). ABT had no effect on Palb in S rats. Acute in vitro incubation of glomeruli isolated from D with ABT (10−6 M, 15 min at 37°C), significantly reduced Palb (0.89±0.11 to 0.41±0.09, P&lt;0.05) while selective ETB blockade, A192621 (10−3 to 10−7 M), had no effect. In conclusion, these data support the hypothesis that ETA but not ETB receptors play an important role in maintaining elevated Palb in type 1 diabetes. Mechanisms responsible for additional changes not affected by ABT remain to be determined.

Effects of early endotoxemia and dextran-induced anaphylaxis on the size selectivity of the glomerular filtration barrier in rats

This study was performed to investigate the glomerular permeability alterations responsible for the microalbuminuria occurring in endotoxemia and during anaphylactic shock. In anesthetized Wistar rats, the left ureter was catheterized for urine collection while, simultaneously, blood access was achieved. Endotoxemia was induced by lipopolysaccharide (LPS) from Escherichia coli, and glomerular permeability was assessed at 60 and 90 (n = 7) and 120 (n = 7) min. Anaphylaxis was induced by a bolus dose of Dextran-70, and glomerular permeability assessed at 5 min (n = 8) and 40 min (n = 9). Sham animals were followed for either 5 or 120 min. The glomerular sieving coefficients (theta) to fluorescein isothiocyanate-Ficoll (70/400) were determined from plasma and urine samples and assessed using size-exclusion chromatography (HPLC). After start of the LPS infusion (2 h), but not at 60 or 90 min, theta for Ficoll(70A) had increased markedly [from 2.91 x 10(-5) +/- 6.33 x 10(-6) to 7.78 x 10(-5) +/- 6.21 x 10(-6) (P < 0.001)]. In anaphylaxis, there was a large increase in theta for Ficolls >60 A in molecular radius already at 5 min, but the glomerular permeability was completely restored at 40 min. In conclusion, there was a transient, immediate increment of glomerular permeability in dextran-induced anaphylaxis, which was completely reversible within 40 min. By contrast, endotoxemia caused an increase in glomerular permeability that was manifest first after 2 h. In both cases, theta to large Ficoll molecules were markedly increased, reflecting an increase in the number of large pores in the glomerular filter.

Urinary excretions of lipocalin-type prostaglandin D2 synthase predict the development of proteinuria and renal injury in OLETF rats

Otsuka Long-Evans Tokushima Fatty (OLETF) rats genetically develop diabetes which is associated with hypertension. In preliminary studies, urinary excretions of L-PGDS (lipocaline-type prostaglandin D synthase) increase before diabetic nephropathy obviously develops, and this may predict progression of renal injury following diabetes. In the present study, we attempted to define whether urinary excretions of L-PGDS behave as the predictor of development of diabetic nephropathy in OLETF rats. We investigated alterations of urinary L-PGDS excretions during the establishment of diabetes and assessed the relationship between the L-PGDS excretions and renal function in OLETF rats. Furthermore, we treated OLETF rats with troglitazone and analysed the effects on L-PGDS metabolisms. Urinary L-PGDS was measured by immunoenzyme assay and the occurrence of L-PGDS and its mRNA in the kidney was assessed by immunohistochemistry and a PCR method. Urinary excretions of L-PGDS were significantly higher in OLETF rats than non-diabetic Long-Evans Tokushima Otsuka (LETO) rats. The excretions age-dependently increased in OLETF and this increase appeared to be due to increased glomerular permeability to L-PGDS. Messenger RNA and antigenicity of L-PGDS were demonstrated in renal tissue; however, the de novo synthesis of L-PGDS mRNA seemingly contributed to urinary L-PGDS excretions much less than glomerular filtration. Multiple regression analysis revealed that urinary L-PGDS was determined by urinary protein excretions, and not by high blood pressure per se. Conversely, urinary proteinuria in the established diabetic nephropathy was predicted by urinary L-PGDS excretions in the early stage of diabetes. Urinary excretions of L-PGDS are likely to reflect the underlying increase in glomerular permeability. This property may be useful to predict forthcoming glomerular damage following diabetes in OLETF rats.